Strain of bacteria of the species Lactobacillus paracasei subsp. paracasei, composition thereof for use in food and product containing said strain

ABSTRACT

Strain of Lactobacillus useful as probiotics in food and naturopathic medicines and which is resistant in vitro against hydrochloric acid and gastric juices and tolerates bile salts without deconjugating them whereas strong assimilation is occurring and which has good survival at the passage through the stomach and the gastrointestinal tract and which strain is growing optimally at about 37° C., which strain is  Lactobacillus paracasei  subsp.  paracasei,  which is a Gram-positive, homofermentative, rod-shaped bacterium capable of producing L-lactic acid and containing three plasmids having a size of 2.2, 4.36 and 9.1 Kb, respectively. The invention also relates to a composition containing the strain and a product consisting of or containing a concentrate of the strain.

This application was filed under 35 USC 371 as the national phase ofPCT/SE98/02263 filed Dec. 8, 1998.

The present invention relates to a strain of Lactobacillus paracaseisubsp. paracasei, a composition thereof for use in food as well as aproduct containing said strain.

Definition and Characterisation of the Strain

The novel strain (which in the following for simplicity will bedesignated LMG-P-17806) is a variant of the species Lactobacillusparacasei subsp. paracasei. It has the characteristics of the specieswith a GC-content of 44%. LMG P-17806 has been isolated from samplesfrom the gastrointestinal micro-flora of humans. LMG-P-17806 is aGram-positive, homofermentative rod-shaped bacteria. It producesL-lactic acid (laevorotatory stereoisomer of lactic acid) and growsoptimally at 37° C. The stain is characterized by being tolerantin-vitro against hydrochloric acid and gastric juice by tolerating bilesalts without deconjugating them and by having a great ability ofassimilating cholesterol. The stain is also characterized by containingthree plasmids having a size of 2.2, 4.36 and 9.1 Kb, respectively.Other characteristics are that the strain is fermenting ribose,adonitol, galactose, glucose, fructose, mannose, sorbose, mannitol,sorbitol, N-acetyl-glucosamine, esculin, cellobiose, maltose, lactose,sucrose, trehalose, inulin, melezitose, D-turanose and D-tagatose. Onthe other hand it does not ferment glycerol, erythritol, D- andL-arabinose, D- and L-xylose, β-methyl-D-xyloside, rhamnose, dulcitol,inositol, α-methyl-D-mannoside, α-methyl-D-glucoside, amygdalin,arbutin, salicin, melibiose, raffinose, starch, glycogen, xylitol,gentiobiose, D-lyxose D- and L-fucose, D- and L-arabitol and 2- and5-ketogluconate.

The strain has been characterized by SDS gel electrophoresis, in whichit has been compared to six other strains of Lactobacillus paracaseisubsp. paracasei, vide the accompanying figure. In this comparison ithas been shown to differ from all other described strains and at thesame time as it when being compared to other lactobacilli appears tobelong to the designated species. It has also been characterized withregard to ribosomic RNA in a so called Riboprinter®. With this methodthe strain has been shown to possess 76% similarity with the type strainfor Lactobacillus paracasei subsp. paracasei and 72% similarity to thetype strain of Lactobacillus casei analyzed at the same occasion.

The strain has been deposited at Belgian Coordinated Collections ofMicroorganisms—BCCM, LMG collection, and there been given the accessionNo. LMG P-17806.

The Advantages of the Strain

LMG P-17806 has, when compared to known strains of Lactobacillus,crucial advantages in the use as probiotics in food and naturopathicmedicines, i.e., medical food, by a unique combination of goodproperties;

the strain has good resistance against gastric juice and bile salts, butunlike many other strains it does not deconjugate the bile salts;

it has a great ability to assimilate cholesterol;

the strain is well managing the passage through the stomach;

the strain has an influence on the conditions in the model of largeintestine by increasing the production of L-lactic acid therein;

the strain is not more pro-inflammatory than common yogurt bacteria;

the strain prevents intestinal cells from being invaded by pathogenicmicroorganisms, such as Salmonella typhmurium;

the strain has an antagonistic action against the gastric ulcerbacterium Helicobacter pylori:

the strain forms bacteriocins which are active against clostridiae;

the strain survives well in milk as well as in frozen and dried form;

the strain, unlike most other lactobacilli has a favourable influence onthe taste of fermented milk products (does not give any tang).

The present strain of Lactobacillus paracasei subsp. paracasei can beused as an additive to food or as naturopathic medicine, so called“Medical Food”, or as an additive to naturopathic medicine.

Such medicines can be used for children with the purpose of alleviatingatopic problems; for elderly persons in order to correct alteredmicroflora caused by normal alterations by age or an altered secretionof hydrochloric acid; and for persons in general in order to normalizethe intestine flora, whereas the content of clostridium bacteria isdecreasing, lactobacilli and bufido bacteria being increased and highcontents of coliformic bacteria being decreased.

By means of these properties the strain LMG P-17806 differs frompreviously known strains, which will be shown in the examples below.

Preparation of the Strain

The strain is prepared in the usual way for lactobacilli. A substratesuited for lactobacilli is used. This substrate should for instancecontain at least one of the carbohydrates which the strain can fermentaccording to what is stated above, in combination with proteins,vitamins, minerals and other nutrients which normally are required bylactobacilli. Examples of suitable commercial substrates are yeastextract-glucose broth, MRS (de Man-Rogosa-Sharp broth), Rogosa, milkadded with a minor amount of a yeast extract, etc. The strain iscultivated microaerophilicly or in the complete absence of oxygen,suitably at a temperature between +15° C. and +42° C. If the substrateis grafted with 0.1 to 1% of graft a culture time of between 10 and 40hours is suitable. The strain can, if desired, be concentrated bycentrifugation or filtration followed by washing the concentrate inorder to remove the culture medium. The concentration can then be frozenor lyophilized in the common way. In this way preparations of between100 millions and 100,000 millions of living bacteria LMG-P-17806 per gcan be prepared. A preparation can then be used as such or be used as anadditive to food, for instance to milk or another product which givesLMG P-17806 the possibility to survive and, if desired, to grow.

Investigations

A. Investigation Concerning the Passage of the Strain Through theGastrointestinal Tract

LMG- P-17806 was cultivated in the way described above and addedtogether with yogurt culture to milk. A fermented product was producedby incubating the milk for five hours at +42° C. A palatable product wasobtained which contained fully 100 millions living LMG P-17806 per gramof product. Healthy persons were given 3×200 g product daily for oneweek. The total intake of LMG P-17806 was between 40 billions and 200billions.

Faeces samples were examined before the intake, after one week ofconsumption and one week after the intake had ceased. As is evident fromTables 1 and 2 below, a strong increase in the number of lactobacilli inthe test subjects was obtained. Two isolates per test subject wereclassified as to species on each occasion, i.e. 20 isolates in total. 18of the isolates for the consumption time appeared to consist of LMGP-17806 according to fenotypical classification. This bacteria strainwas not discovered in the samples before or after the intake of LMGP-17806.

In average the contents of the faeces samples during the supply werevery high and varied only moderately from 63 millions to 320 millionsper gram, i.e. with a factor of 5. Noteworthy was, that the contentswere largely the same independent of what contents were measured beforethe start of the experiment. After the supply had ceased the contentsreverted to what seems to be natural for the test subject in question.

Tables 1 and 2 below the content of lactobacilli in faeces determined,in millions per gram, by plating and using the substrate Rogosa.

TABLE 1 5 test subjects with originally low content of lactobacilli TestBefore the During the After the subject experiment experiment experiment1 0.05 120 0.07 2 0.15 320 0.12 3 0.08 97 0.14 4 0.009 63 0.02 5 <0.001278 0.002

TABLE 2 5 test subjects with high contents of lactobacilli Test Beforethe During the After the subject experiment experiment experiment 6 1.2297 1.3 7 0.7 83 0.6 8 0.2 136 0.18 9 4.3 74 3.5 10 0.6 212 0.8

The examination shows that the strain has good survival at the passagethrough the gastrointestinal tract.

B. Examination Concerning the Formation of L-lactic Acid by the Strainin a Model of Large Intestine

The fermented product above was added to a so called SHIME-reactor whichis an in vitro model of the intestine. Samples were taken from the partof the reactor which corresponds to the most important parts of thelarge intestine. Similar comparative tests were carried out with someother Lacto bacillus strains, i.a. closely related L. paracasei subsp.paracasei. As is evident from the following Table 3 below LMG P-17806gave a strong increase in the production of L-lactic acid, which is thevery lactic acid isomer which is generated by LMG P-17806. A productionof lactic acid is considered as favourable for several reasons, i.a.considering the anti-bacterial effect of the lactic acid as well as thefact that a lower pH is supposed to reduce the availability andformation of nitrogen compounds.

TABLE 3 Production of L- and D-lactic acid in a SHIME-reactor after theaddition of different lactic acid bacteria in mg per litre reactorcontent Lactic acid Reactor 4 Reactor 5 Reactor 6 bacteria L D L D L DLMG P-17306 300 80 500 100 280 90 L. paracasei 40 80 200 70 130 40 L.rhamnosus 90 10 10 70 80 10 L. plantarum 60 50 60 70 40 50

In the table “L” refers to the laevorotatory isomer of lactic acid and“D” to the dextrorotatory isomer.

Reactor 4 corresponds to the upper part of the large intestine, reactor5 to the middle part and reactor 6 to the lower part of the largeintestine. The investigation shows that the strain is forming L-lacticacid in the model of the large intestine.

C. Investigation of How the Strain is Protecting Intestinal EpitheliumCells from Invasion of Salmonella Typhimurium

Intestinal epithelium cells of the type CaCo-2 cells were cultivatedin-vitro. These were added with a combination of lactobacilli andSalmonella typhimurium in the ratio 100:1 with the addition of 1 millionsalmonella per ml. The effect was studied after incubation for 120 minat 370° C. The amount of invading salmonella was determined by washingthe plates with adhering CaCo-2 cells three times. The adhering cellswere treated with the antibioticum gentamycin in a concentration of 100mg/l for one hour in order to kill all bacteria which had not invadedcells. Then the plates were washed with PBS in order to remove allgentamycin and finally the entrapped bacteria were released by treatingthe CaCo-2 cells with 0.1% Triton-X during shaking. The number ofsalmonella was then determined by common plating methodology. LMGP-17806 had a pronounced effect in that it reduced a number of invadedcells. The closely related paracasei-variant 506 on the other handseemed rather to stimulate the invasion of salmonella bacteria. Alsowith regard to this property LMG P-17806 showed a positive effect. Theresults are reported in Table 4 below.

TABLE 4 Invasion of salmonella % invading salmonella without lactic acidwith lactic acid Lactic acid bacteria bacteria bacteria LNG P-17806 2.50.75 L. paracaseiK 506 2.5 7 L. plantarium 2 2 L. rhamnosus 2.5 0.75

The table shows that the strain LMG P-17806 gives a marked protectionagainst invasion of salmonella bacteria.

D. Investigation of Protection Against Helicobacter Pylori

In a mouse model where the mice had been infected with Helicobacterpylori the effect of supplying a fermented milk product with a strain ofLMG P-17806 on one hand and without said strain on the other on thecontent of H. pylori measured in faeces was examined.

The mice were infected with 100 millions of the strain H. Pylori 17874in helical form at three occasions with an interval of one day. Then themice were given experimental products and the content of H. pylori infaeces was measured by means of heparinised magnetic balls and EnzymeImmuno Assay. Three products were examined. All the fermented milkproducts appeared to reduce the share of H. pylori, but the effect wasoccurring considerably faster in the cases when the product containedLMG P-17806 in comparison to common yoghurt and in comparison with astrain of L. fermentum KLD, respectively.

TABLE 5 Content of H. pylori in faeces measured by heparinised magneticballs and Enzyme Immuno Assay after after 7 days before intake forintake for after cease Product intake 2 days 7 days of intake Commonyoghurt 1.48 1.86 0.63 2.25 Yoghurt with 1.65 1.62 0.94 1.61 L.Fermentum Yoghurt with 1.80 0.68 0.61 1.71 LMG P-17806

The figures in the table state the absorbency of 405 nm and are relativecontents.

E. Examination of the Influence of the Strain LMG P-17806 on theImmunological Defence

The immunological defence system is controlled by a series of signalsubstances, so called cytokins. Some of these can be proinflammatory.The influence of LMG P-17806 on the production of cytokins TNF-alfa andIL-6 was compared with the influence of the two species contained in ayoghurt culture, L. delbruckei subsp. bulgaricus and Streptococcusthermophilus. Leucocytes were separated from human blood and added toliving bacteria or bacteria killed with glutaraldehyde in an amount of10 millions of leucocytes. As control lipopolysacharides (LPS) from E.coli were used. The results are reported in Table 6 below. The resultsshow that the LMG P-17806 has the same inflammatory properties as acommon yoghurt culture in the model used.

TABLE 6 The influence of the strain LMG P-17806 on the immunologicaldefence TNF-alpha mg/l IL-6 mg/l Lactic acid bacteria living killedliving killed S. thermophilus, E584 12 1 0.4 <0.1 L. bulgaricus, E535 23 0.5 0.1 LMG P-17806 4 1 0.7 <0.1

F. Examination of the Resistance of the Strain LMG P-17806 AgainstAntibiotics

Probiotics can be useful for use in connection with disorders in thebalance of the intestine flora during medication with antibiotics. Atthe same time it is important that probiotics do not contribute tospreading of resistance to antibiotics, and this is especially importantin the contemporary use of probiotics and antibiotics. The resistance ofthe strain LMG P-17806 against different antibiotics has beenestablished for that reason. The sensitivity of the strain LMG P-17806to different antibiotics were determined by establishing the content atwhich a reduction in the growth of the strain by 50%, measured asoptical density, was obtained.

It appeared that the strain LMG P-17806 was resistant against vancomycinand was not inhibited even by 256 mg/l. The strain showed someresistance against trimethoprim and cefotaxime, an optical density (OD)of 50% at 12 and 4 mg/l, respectively being obtained. The strain LMGP-17806 was on the other hand sensitive to chloraphenicol, erythromcyin,rifampicin and tetracycline, where already levels below 1 mg/l resultedin an inhibition of the growth by 50%.

The influence by antibiotics was also examined as a survey by using socalled Sensi discs from Oxoid. According to these results LMG P-17806was resistant against aztreonam, ceftaximid, cefoxitin, colistinesulphate, kanamycin, polymyxin B, streptomycin and vancomycin.

LMG P-17806 might thus according to these results be especiallyinteresting for use at the same time as therapy with antibiotics such asvancomycin, trimethoprim as well as several cefloxacins, which all areantibiotics with known side effects on the intestine flora and intestinefunction.

The Use of the Strain LMG P-17806

EXAMPLE 1 Preparation of Bacterial Concentrate

A frozen bacterial concentrate with 10 billions of LMG P-17806 per gramwas prepared in the way stated above by cultivating the lactobacilli ina substrate of whey added with 1 g yeast extract per liter at a constantpH of 5.5 for 14 hours at 36° C. The bacteria were separated bycentrifugation with continuous washing of the centrifugate. Theconcentrate was frozen in liquid nitrogen and then stored at −80° C.until use.

EXAMPLE 2 Preparation of Fermented Milk Product

A product milk was prepared from milk by homogenizing the milk,heat-treating it at +95° C. for five minutes and tempering it to +37° C.The product milk was grafted with 0.01% of a commercial, frozen yogurtculture and 0.5% of the LMG P-17806 concentrate. The cultures wereallowed to grow for six hours at the temperature stated. The milk hadthen coagulated and the pH decreased to 4.55. The coagulated form wasbroken and the product chilled to +12° C. and then it was packed incommon plastic cups, which are normally used for yogurt, andafter-cooled in a refrigerating chamber having a temperature of +5° C.for one day and night. pH had then decreased to 4.4. The product wasthen stored at +8° C. for up to three weeks.

The content of the strain LMG P-17806 was monitored and as is seen fromthe results in Table 7 below, there was a limited growth of the strainLMG P-17806 during the culture and LMG P-17806 survived storing forthree weeks at pH below 4.4 very well.

TABLE 7 The storability of the strain LMG P-17806 in milk productContent After After After in Content storing storing storing productafter for 1 for 2 for 3 Graft milk cultivation week weeks weeks 9900 4997 132 118 121

The contents are given in millions per gram.

The product had a normal appearance with a separation of whey of barely1% after storing for fourteen days. The product tasted excellent and haduniform consistency and a fresh, mild flavor. The test product receivedbetter judgements than normal yogurt in an independent consumer surveycarried out by an research institute. The product was compared in aconsumer survey, in which the testing persons did not know what productwas tasted, with a corresponding product without the strain LMG P-17806.The product with LMG P-17806 was preferred by 74% of the persons of thetesting panel, and it received the average value of 7.6 in a scale of 9points, which is significantly higher than the result for common yogurt.The judgement was slightly more than 1 point higher than previouslyknown probiotic culture with Lactobacillus acidophilus. The results alsodiffer from those previously obtained in comparison between pure yogurtand mixed products of yogurt and the probiotic bacteria Lactobacillusacidophilus and Bifidobacterium longum, respectively. No significantdifferences could be noted between pure yogurt and the respective mixedproduct in these comparisons.

EXAMPLE 3 Suitable Addition of the Strain LMG P-17806

Experiments were also carried out in order to find out the suitablerange for the addition of the strain LMG P-17806. Milk was treated andthen added with yogurt culture according to the above, whereas theaddition of concentrate of the strain LMG P-17806 was varied from 5% to0.01%. At such a high content as 5% graft of LMG P-17806 a tang wasobtained, which probably originated from components of the graft itself.The change in pH was normal, however, and the appearance of the productwas normal. The content of LMG P-17806 in fresh product was 504 millionsper gram and the contents remained at this level during the storage. Atthe addition of 0.01% LMG P-17806 concentrate the product could notsensoricly be distinguished from common yogurt and the content of LMGP-17806 was already after one week less than one million per ml, whichis the lowest content a product must contain in order to be allowed tostate the product to contain a specific probiotic according to proposalto international regulation.

EXAMPLE 4 Preparation on Fermented Special Product

The experiment was carried out as above except that the milk also wasadded with 0.4 g yeast extract per liter. The graft was performed with0.01% of the same yogurt culture as above, but only 0.1% LMG P-17806concentrate was added. Incubation was carried out at +34° C. for 8 hoursand then the product chilled, packed and stored as in Example 3 above.LMG P-17806 grew ten times under these conditions and similarly to theabove the bacteria survived well during storage. The results arereported in the Table below.

TABLE 8 The storability of the specific milk product of the strain LMGP-17806 Content Content After After After in milk after storing forstoring storing Graft product cultivation one week for two weeks forthree weeks 9900 10 103 114 109 107

The contents in the table are given in millions per gram product.

The product had a normal appearance with a separation of whey of 1.5%after storing for fourteen days. The product had a good, dry flavor andhomogeneous consistency.

EXAMPLE 5 Preparation of Vegetable Juice Containing the Strain LMGP-17806

A vegetable juice was prepared by mixing carrot concentrate and anorange juice concentrate in equal parts so that pH of the finishedmixture became pH 3.9. The mixed beverage was heat-treated and addedwith 1% and 0.1%, respectively, of LMG P-17806 concentrate. The contentof LMG P-17806 became 100 and 10 millions per ml, respectively. Thebeverages were stored at +7° C. for four weeks. The flavor of theproduct was not affected and no influence by storing or addition of LMGP-17806 was observed. Addition of LMG P-17806 to a content exceeding 50millions, however, seemed to reduce the decrease in vitamin C. In aproduct without LMG P-17806, like in the product having 10 millionsbacteria per ml, the content of vitamin C decreased from 25 mg/100 g,before the storage to 18 mg/100 g after four weeks. With the addition of100 millions LMG P-17806 ml the content of vitamin C only decreased to22 mg/100 g, i.e., more than 50% lower decrease in the content ofvitamin C. LMG P-17806 survived well and the content of LMG P-17806after four weeks was only about 40% lower than in fresh productindependent of the amount added.

In another experiment the same fruit beverage was added with 5% LMGP-17806. This gave a tang, which grew worse during storage time.

EXAMPLE 6 Preparation of Cereal Powder Based Food Product

Cereal powder based food product is manufactured in the common way. LMGP-17806 concentrate, according to the above is lyophilized after mixingwith corn starch and stored after being packed in an oxygen-tightwrapping at a temperature of −20° C., The lyophilized preparationcontained 61 billions LMG P-17806 per gram. The cereal was dried-mixedwith 0.03% LMG P-17806 and packed in an atmosphere of nitrogen gas in anoxygen tight wrapping. The cereal was stored initially for three monthsat +12° C. and thereafter at a room temperature for additional fourmonths. The content of LMG P-17806 is apparent from Table 9 below. Thecereal gave good possibilities for survival to LMG P-17806 and thereforeit is possible to prepare, for instance, baby food products having ahigh content of LMG P-17806.

TABLE 9 Lyophilized Cereal before Cereal after After 3 After 7preparation addition addition months months 61,000 <0.0001 20 18 6.5

The content in the Table 9 are given in millions per gram cereal.

The cereal was dissolved in 9 parts of water of +50° C. and the contentof LMG P-17806 determined. The content was 1 million LMG P-17806 per mlbeverage in the case that the cereal was formulated as a six months' oldfood product. The cereal could not be distinguished from common product.After storing for one night in room temperature, however, the cerealadded with LMG P-17806 tasted slightly acid and had pH of 5.7.

In a second experiment the effect of the addition of 1% and 0.001%,respectively, of lyophilized LMG P-17806 preparation was examined. Thecontents thereof in the cereal after the addition were 570 and 0.8millions per gram respectively.

The cereals were stored in a corresponding way and examined afterstorage for 5 months. After dissolution as above the content in theprepared cereal was 800 millions, respectively for the low addition lessthan 10,000 per ml. With the high addition a slightly acid flavor wasnoted already after storing for four hours of the cereal at bodytemperature.

EXAMPLE 7 Preparation of Dried Power for Use as “Medical Food”

A lyophilized LMG P-17806 concentrate was mixed with different amountsof corn starch in the proportions in 1:1, 1:9, 1:99 and 1:999,respectively. The mixed powders were stored in small sachets with 1 to100 g per sachet. The material of the sachets was impervious to oxygenand water vapor. The sachets were stored in freezer, refrigerator androom temperature, respectively. The content was then used as an additiveto beverage by mixing the powder with the beverage before the beveragebeing consumed. The corresponding survival as in example 6 above wasobtained. The powder with the proportions 1:1 was also packed in gelatincapsules with 0.4 g per capsule. The number of bacteria per capsule was10 billions. The capsules were blister-packed in a material with goodbarrier properties against oxygen and water vapor. The capsules werestored in the same way as stated above and corresponding good survivalresults were obtained.

EXAMPLE 8 Preparation and Use of Bacterial Mixes

LMG P-17806 can be mixed with other lactobacilli without inhibitingthem. LMG-17806 does not seem to form any substances which areinhibiting other lactobacilli or lactococci where LMG P-17806 areco-cultivated with yogurt cultures, sour milk cultures with lactococcior other lactobacilli species such as L. acidophilus, L. fermentum or L.rhamnosus. Experiments with mixtures of lyophilized preparationscontaining all these bacteria have indicated unchanged storageproperties whether mixing was carried out with corn starch or with pappowder. After the solution of the pap powder and a storage for 12 hoursat body temperature and 12 hours at room temperature no negativeinfluence by LMG P-17806 could be traced either on the total content oflactobacilli or on the content of either of the bacteria.

In an experiment to produce probiotic sour milk 0.5% of sour milkculture and 0.6% of lyophilized concentrate of each of L. acidophilusNCFB 1748, L. fermentum KLD and LMG p-17806 were added to normal treatedproduct milk. However, only 0.1% KLD was added, because higher additionsgave rise to tang and to a bad coagulum. Milk was stored for 19 hours atroom temperature. Then the pH thereof was 4.50. The milk was cooled to+10° C., packed and stored at +6° C. or up to 14 days. The results areevident from the following table 10.

TABLE 10 Lactic acid bacteria Before cooling After 5 days After 14 daysLactococci 730 810 580 L. acidophilus 135 142 131 L. fermentum 12 14 9LMG P-17806 112 126 109

The contents are in millions per ml of the different bacteria.

80 healthy test subjects were given the products to eat in connectionwith a conference journey to Istanbul in Turkey. The group was dividedinto two, one eating a sour milk according to the above with only LMGP-17806 whereas the other ate the probiotic sour milk with threedifferent probiotic strains. The test subjects were on the conferenceplace for 8 days. They started to eat the products two days beforedeparture to the conference place and continued to eat for four daysafter home-coming. The products were eaten as 3 snacks evenly spreadduring the day with 150 g/meal. All test subjects except 2 persons ofthe sour milk group declared that they ate the product according to thescheme. In an inquiry they were asked to state discomforts from thegastrointestinal tract in the form of stomach pains, tensions, diarrhoeaor constipation on a scale of 3 degrees. Apart from diarrhoea there wasa difference so far that 5% of the test subjects which only had eatenLMG P-17806 stated that they had had serious or very serious diarrhoeaduring at least two days whereas this frequency only was 22% in thegroup which ate the probiotic sour milk.

Thus it seems as if a mix of several different lactobacilli might bestill more effective than only one single strain of bacteria. This canbe due to the fact that the lactobacilli administered had differentproperties.

What is claimed is:
 1. An isolated strain of Lactobacillus paracaseisubsp. paracasei, LMG-P-17806, said strain being a Gram-positive,homofermentative, rod-shaped bacterium capable of producing L-lacticacid and containing three plasmids having a size of 2.2, 4.36 and 9.1Kb, respectively.
 2. The strain according to claim 1, wherein the straincontains 44% GC.
 3. The strain according to claim 1, wherein the strainhas been isolated from samples from the gastrointestinal micro-flora ofhumans.
 4. The strain according to claim 1, wherein the strain canferment ribose, adonitol, galactose, glucose, fructose, mannose,sorbose, mannitol, sorbitol, N-acetyl-glucosamine, esculin, cellobiose,maltose, lactose, sucrose, trehalose, inulin, melezitose, D-turanose andD-tagatose.
 5. The strain according to claim 1, wherein the strain isprovided as a concentrate in the form of a frozen or lyophilized powder.6. A method for producing food products, said method comprising: addingLactobacillus paracasei subsp. paracasei, LMG P-17806 to a food productselected from the group consisting of a milk product, a fermented milkproduct, a powdered cereal based food, a food for infants, a food forsmall children, a fruit beverage and a vegetable beverage, in an amountof between 0.001% and 5% of the food product.
 7. The method of claim 6wherein the strain is present in an amount of between 1 million and 10billion living bacteria per gram of the product.
 8. The method of claim6, wherein the strain is added in a content of between 0.01% and 1%. 9.A method for cultivating the strain of Lactobacillus paracasei subsp.paracasei, LMG P-17806, said method comprising: growing the straintogether with other lactic acid bacteria used in the production offermented food products.
 10. The method of claim 7 wherein the strain ispresent in an amount of between 1 million and 10 billion living bacteriaper gram of the food.
 11. A composition containing the strainLactobacillus paracasei subsp. paracasei, LMG P-17806, said compositioncomprising: a food product selected from the group consisting of a milkproduct, a fermented milk product, a vegetable beverage, a fruitbeverage, a powdered cereal based product, a food for infants, and afood for small children, said food product containing the strainLactobacillus paracasei subsp. paracasei in an amount of 5×10⁵-5×10⁹living bacteria, corresponding to 0.0005-0.5% of the food product. 12.The composition according to claim 11 wherein the food product furthercomprises a yeast extract or a nutrient which contributes to growth orsurvival of Lactobacillus paracasei subsp. paracasei in the foodproduct.
 13. The product of claim 12, wherein the yeast extract ispresent in an amount between 0.01 and 0.1%.
 14. The product according toclaim 12, wherein the strain is in the form of a concentrate in a frozenor a lyophilized condition for mixing in direct connection withconsumption occasion into the food product.
 15. The compositionaccording to claim 11, wherein the strain is present in the form of aconcentrate in a frozen or a lyophilized condition for mixing at thetime of consumption of the food product.
 16. The product of claim 11wherein the strain is in an amount of 1×10⁵-10⁹ living bacteria.
 17. Afood product comprising: a beverage selected from the group consistingof a milk product, a fermented milk product, a fruit beverage and avegetable beverage; and the strain of Lactobacillus paracasei subsp.paracasei, LMG P-17806 present in said beverage in an amount of between0.001% and 5%.
 18. The food product of claim 17, wherein the strain ispresent in an amount of between 0.01% and 1%.
 19. The food product ofclaim 18 wherein the strain is present in an amount of between 1 millionand 10 billion living bacteria per gram of the food.
 20. The foodproduct of claim 17 wherein the strain is present in an amount ofbetween 1 million and 10 billion living bacteria per gram of the food.21. A composition containing the strain Lactobacillus paracasei subsp.paracasei, LMG P-17806, said composition comprising: a concentratednutritional supplement wherein the strain Lactobacillus paracasei subsp.paracasei is present at a content of 1-100 billion living bacteriacorresponding to 0.001-100% of the concentrated nutritional supplement.22. The composition according to claim 21 wherein the nutritionalsupplement further comprises a nutrient for enhancing survival of thebacteria.
 23. The composition according to claim 22, wherein the strainis in the form of a concentrate in a frozen or a lyophilized conditionfor mixing in direct connection with consumption occasion into thenutritional supplement.
 24. The composition according to claim 21,wherein the strain is present in the form of a concentrate in a frozenor a lyophilized condition for mixing at the time of consumption of thenutritional supplement.